Recent pharmacological and genetic data underscore the importance of the EGF receptor (EGFR) axis in the pathogenesis of premalignant and malignant disorders within the gastrointestinal tract. An additional emerging theme is that proteolytic cleavage of EGFR ligands is a critical step in regulation of EGFR signaling. Combined blockade of EGFR binding and TGFalpha and amphiregulin (AR) cleavage cooperate to inhibit growth of colon cancer cells in vitro. Collectively, these data indicate the tractability of the EGFR axis as a therapeutic target for premalignant and malignant human gastrointestinal disorders. A major focus during the previous granting period was to explore the trafficking and processing of EGF, TGFalpha and AR in the context of polarized epithelial cells. Fundamental insights have emerged from these in vitro polarizing epithelial model systems that have contributed to the advances cited above. For example, TGFalpha is a locally captured growth factor whereas the actions of AR are more complex, but both ligands are cleaved at the basolateral surface by TNF-alpha converting enzyme (TACE). In a comprehensive series of experiments focused around a central theme (role of EGFR axis in colonic neoplasia) and framed as a set of testable hypotheses, we propose the following specific aims: 1. Advance our basic studies of EGFR axis regulation in polarized epithelial cells in vitro. Studies will include regulation of TGFalpha and AR cleavage, distinct actions of AR mediated by its interactions with heparan sulfate proteoglycans, and biological roles for two novel genes that bind the cytoplasmic tail domain of TGFalpha. 2. Examine selected components of the EGFR axis as therapeutic targets in colon cancer (EGFR ligand binding, EGFR tyrosine kinase activity, and TACE cleavage of TGFalpha and AR); 3. Determine precise roles for TGFalpha and AR in the pathogenesis of colonic neoplasia by two complementry approaches in the mouse: targeted overexpression of TGFalpha and AR in the gut; effect of targeted disruption of TGFalpha and AR in spontaneous and carcinogen-induced intestinal tumorigenesis.